HGF-mediated chemotaxis and tubulogenesis require activation of the phosphatidylinositol 3-kinase

1995 ◽  
Vol 268 (6) ◽  
pp. F1211-F1217 ◽  
Author(s):  
M. P. Derman ◽  
M. J. Cunha ◽  
E. J. Barros ◽  
S. K. Nigam ◽  
L. G. Cantley
Keyword(s):  
Collecting Duct ◽  
Collagen Matrix ◽  
Phosphatidylinositol 3 Kinase ◽  
Process Formation ◽  
High Affinity Receptor ◽  
Collecting Duct Cells ◽  
Medullary Collecting Duct ◽  
Phosphatidylinositol 3

The association of hepatocyte growth factor (HGF) with its high-affinity receptor, c-met, has been shown to induce mitogenesis, motogenesis, and morphogenesis in renal epithelial cells (L. G. Cantley, E. J. G. Barros, M. Gandhi, M. Rauchman, and S. K. Nigam. Am. J. Physiol. 267 (Renal Fluid Electrolyte Physiol. 36): F271-F280, 1994), suggesting that HGF may be critical to the orchestration of both renal development and regeneration following injury. Although signal transduction pathways activated by c-met include the phosphatidylinositol 3-kinase (PI-3-kinase), phospholipase C gamma, ras, and others, the activation of PI-3-kinase has been the most striking in vivo. We therefore investigated whether the pathways that mediate phenotypic changes in inner medullary collecting duct cells are altered by inhibition of PI-3-kinase with the fungal metabolite, wortmannin. In these cells, the mean inhibitory concentration for in vitro wortmannin inhibition of PI-3-kinase was approximately 0.2 nM. At this low concentration, motogenesis (quantified by chemotaxis) and morphogenesis (by branching-process formation within collagen matrix) were inhibited in a striking and parallel fashion, while mitogenesis was inhibited to a lesser degree. These experiments suggest that activation of PI-3-kinase is critical for c-met-mediated chemotaxis and tubulogenesis.

Acidification adaptation in cultured inner medullary collecting duct cells

1993 ◽  
Vol 264 (5) ◽  
pp. F765-F769 ◽  
Author(s):  
R. Mankus ◽  
J. H. Schwartz ◽  
E. A. Alexander
Keyword(s):  
Lucifer Yellow ◽  
Collecting Duct ◽  
C Cells ◽  
Inner Medullary Collecting Duct ◽  
Pump Activity ◽  
Collecting Duct Cells ◽  
Medullary Collecting Duct ◽  
New Protein

Chronic acid feeding stimulates the rat inner medullary collecting duct (IMCD) to increase acid secretion in vivo (acidification adaptation), but the mechanism for this phenomenon is unknown. Our purpose was to determine whether IMCD cells undergo adaptation in vitro and to explore the mechanism of this response. Confluent cultured rat IMCD cells were exposed to incubation media supplemented with 10(-7) M deoxycorticosterone acetate, pH 7.0 [acid incubated (AI)] or 7.7 [control (C)], for 48 h, and cell pH (pHi) was determined using 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. Resting pHi was 7.46 +/- 0.05 for AI and 7.25 +/- 0.04 for C (P < 0.05). N-ethylmaleimide-sensitive pHi recovery after an acute acid pulse was 0.030 +/- 0.002 for AI and 0.020 +/- 0.002 pH U/min for C (P < 0.05). However, when AI and C cells were incubated with 7 x 10(-6) M cycloheximide, the increment in pHi and enhanced proton pump activity was abolished. In addition, exocytic function, as measured by Lucifer yellow release, was increased significantly in AI cells. In summary, incubation of IMCD cells in acid medium stimulates acidification adaptation by a mechanism dependent on new protein synthesis.

Is the function of the renal papilla coupled exclusively to an anaerobic pattern of metabolism?

1979 ◽  
Vol 236 (5) ◽  
pp. F423-F433 ◽  
Author(s):  
J. J. Cohen
Keyword(s):  
Oxidative Metabolism ◽  
Aerobic Glycolysis ◽  
Collecting Duct ◽  
High Rate ◽  
O2 Uptake ◽  
Duct Cells ◽  
Collecting Duct Cells ◽  
Mitochondrial Oxidative Metabolism

It is widely accepted that in vivo the function of the papilla of the mammalian kidney is supported primarily by anaerobic metabolism. As a result, the major source of energy for support of function in the papilla is considered to be derived from glycolysis. This orientation originates from two concepts: 1) that in vivo the gaseous environment of the papilla has such a low PO2 that O2 availability limits O2 consumption, and 2) that papillary tissue has a high rate of glycolysis when compared with either cortical tissue or extrarenal tissues. It has also been tacitly assumed that papillary tissue has a "low" O2 uptake. Review of the measurements of PO2 of papillary tissue and of urine PO2 indicates that the PO2 of papillary tissue should not limit its aerobic mitochondrial oxidative metabolism. While the rate of aerobic glycolysis in papillary tissue is high, simultaneously papillary tissue has a rate of O2 uptake similar to that of liver and higher than that of muscle. The major (two-thirds) source of energy for papillary tissue in vitro is from O2 uptake. That papillary tissue is not exclusively dependent on glucose for its energy requirements is indicated by the greater stimulation of papillary tissue QO2 by succinate than by glucose. Thus, papillary tissue has both a high aerobic mitochondrial oxidative metabolism and a high aerobic glycolytic metabolism. It is suggested that the mechanism for the high rate of aerobic glycolysis in the presence of an adequate O2 supply is due to the relatively small mass of mitochondria in papillary tissue in relation to the amount of work done by the tissue. As a result of the limited rate of ATP production by the mitochondrial electron transport chain, the phosphorylation state ([ATP]/[ADP][Pi]) is reduced and the cytoplasmic redox state ([NAD+]/[NADH]) of the papillary collecting duct cells also becomes more reduced; changes in both ratios enhance the rate of glycolysis. This limited metabolic capacity of the collecting duct cells may permit an excess volume of solute and water to be excreted during volume expansion diuresis. The metabolic characteristics of the papilla, when compared to cortex, also provide a basis for the observed differences in substrate selectivity of cortex and medulla with respect to utilization of glucose and lactate. The experimental approaches that may provide information bearing on the suggested mechanisms for regulation of papillary metabolism in relation to tubular work functions are indicated.

scholarly journals Tyrosine 1101 of Tie2 Is the Major Site of Association of p85 and Is Required for Activation of Phosphatidylinositol 3-Kinase and Akt

1998 ◽  
Vol 18 (7) ◽  
pp. 4131-4140 ◽  
Author(s):  
Christopher D. Kontos ◽  
Thomas P. Stauffer ◽  
Wen-Pin Yang ◽  
John D. York ◽  
Liwen Huang ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Vascular Development ◽  
Pi3 Kinase ◽  
Pleckstrin Homology Domain ◽  
Phosphatidylinositol 3 Kinase ◽  
Lipid Kinase ◽  
Embryonic Vascular Development ◽  
Phosphatidylinositol 3

ABSTRACT Tie2 is an endothelium-specific receptor tyrosine kinase that is required for both normal embryonic vascular development and tumor angiogenesis and is thought to play a role in vascular maintenance. However, the signaling pathways responsible for the function of Tie2 remain unknown. In this report, we demonstrate that the p85 subunit of phosphatidylinositol 3-kinase (PI3-kinase) associates with Tie2 and that this association confers functional lipid kinase activity. Mutation of tyrosine 1101 of Tie2 abrogated p85 association both in vitro and in vivo in yeast. Tie2 was found to activate PI3-kinase in vivo as demonstrated by direct measurement of increases in cellular phosphatidylinositol 3-phosphate and phosphatidylinositol 3,4-bisphosphate, by plasma membrane translocation of a green fluorescent protein-Akt pleckstrin homology domain fusion protein, and by downstream activation of the Akt kinase. Activation of PI3-kinase was abrogated in these assays by mutation of Y1101 to phenylalanine, consistent with a requirement for this residue for p85 association with Tie2. These results suggest that activation of PI3-kinase and Akt may in part account for Tie2’s role in both embryonic vascular development and pathologic angiogenesis, and they are consistent with a role for Tie2 in endothelial cell survival.

Urodilatin: binding properties and stimulation of cGMP generation in rat kidney cells

1993 ◽  
Vol 264 (2) ◽  
pp. F267-F273
Author(s):  
H. Saxenhofer ◽  
W. R. Fitzgibbon ◽  
R. V. Paul
Keyword(s):  
Guanylate Cyclase ◽  
Mesangial Cells ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Binding Characteristics ◽  
Papillary Collecting Duct ◽  
Collecting Duct Cells ◽  
Medullary Collecting Duct ◽  
Anp Receptors

Urodilatin (URO) [ANP-(95-126)] is an analogue of atrial natriuretic peptide (alpha-ANP) [ANP-(99-126)] that was first isolated from human urine. In rat mesangial cells, URO competed with high affinity for non-guanylate cyclase-coupled ANPR-C receptors [concentration at which 50% labeled ligand is displaced (IC50) approximately 70 pM], but with lesser affinity to the guanylate cyclase-linked ANPR-A receptors (IC50 approximately 800 pM). alpha-ANP bound to both receptors with similar affinity [dissociation constant (Kd) approximately 150 pM]. In papillary collecting duct homogenates, which possess only ANPR-A receptors, the apparent Kd value averaged 229 pM for alpha-ANP and 2.7 nM for URO. Intravenous URO was at least as potent and effective as alpha-ANP in inducing diuresis and natriuresis in anesthetized rats, but URO was approximately 10-fold less potent in stimulating guanosine 3',5'-cyclic monophosphate generation in mesangial and inner medullary collecting duct cells. We conclude that URO has a lesser affinity than alpha-ANP for guanylate cyclase-coupled ANP receptors in the kidney and that the relative natriuretic potency of URO in vivo cannot be directly attributed to its binding characteristics with ANPR-A receptors.

scholarly journals 11C-Labeled Pictilisib (GDC-0941) as a Molecular Tracer Targeting Phosphatidylinositol 3-Kinase (PI3K) for Breast Cancer Imaging

2019 ◽  
Vol 2019 ◽  
pp. 1-11 ◽  
Author(s):  
Na Han ◽  
Yaqun Jiang ◽  
Yongkang Gai ◽  
Qingyao Liu ◽  
Lujie Yuan ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Pik3ca Mutation ◽  
Phosphatidylinositol 3 Kinase ◽  
Pet Tracer ◽  
Positron Emission ◽  
Pet Scans ◽  
Mcf 7 ◽  
Phosphatidylinositol 3

Pictilisib (GDC-0941) is an inhibitor of phosphatidylinositol 3-kinase (PI3K), part of a signaling cascade involved in breast cancer development. The purpose of this study was to evaluate the pharmacokinetics of pictilisib noninvasively by radiolabeling it with 11C and to assess the usability of the resulting [11C]-pictilisib as a positron-emission tomography (PET) tracer to screen for pictilisib-sensitive tumors. In this study, pictilisib was radiolabeled with [11C]-methyl iodide to obtain 11C-methylated pictilisib ([11C]-pictilisib) using an automated synthesis module with a high radiolabeling yield. Considerably higher uptake ratios were observed in MCF-7 (PIK3CA mutation, pictilisib-sensitive) cells than those in MDA-MB-231 (PIK3CA wild-type, pictilisib-insensitive) cells at all evaluated time points, indicating good in vitro binding of [11C]-pictilisib. Dynamic micro-PET scans in mice and biodistribution results showed that [11C]-pictilisib was mainly excreted via the hepatobiliary tract into the intestines. MCF-7 xenografts could be clearly visualized on the static micro-PET scans, while MDA-MB-231 tumors could not. Biodistribution results of two xenograft models showed significantly higher uptake and tumor-to-muscle ratios in the MCF-7 xenografts than those in MDA-MB-231 xenografts, exhibiting high in vivo targeting specificity. In conclusion, [11C]-pictilisib was first successfully prepared, and it exhibited good potential to identify pictilisib-sensitive tumors noninvasively, which may have a great impact in the treatment of cancers with an overactive PI3K/Akt/mTOR signal pathway. However, the high activity in hepatobiliary system and intestines needs to be addressed.

Electrolyte and fluid secretion by cultured human inner medullary collecting duct cells

2002 ◽  
Vol 283 (6) ◽  
pp. F1337-F1350 ◽  
Author(s):  
Darren P. Wallace ◽  
Marcy Christensen ◽  
Gail Reif ◽  
Franck Belibi ◽  
Brantley Thrasher ◽  
...  
Keyword(s):  
Fluid Transport ◽  
Collecting Duct ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Fluid Secretion ◽  
Transport Studies ◽  
Collecting Ducts ◽  
Collecting Duct Cells ◽  
Medullary Collecting Duct

Inner medullary collecting ducts (IMCD) are the final nephron segments through which urine flows. To investigate epithelial ion transport in human IMCD, we established primary cell cultures from initial (hIMCDi) and terminal (hIMCDt) inner medullary regions of human kidneys. AVP, PGE2, and forskolin increased cAMP in both hIMCDi and hIMCDt cells. The effects of AVP and PGE2 were greatest in hIMCDi; however, forskolin increased cAMP to the same extent in hIMCDi and hIMCDt. Basal short-circuit current ( I SC) of hIMCDi monolayers was 1.4 ± 0.5 μA/cm2 and was inhibited by benzamil, a Na+ channel blocker. 8-Bromo-cAMP, AVP, PGE2, and forskolin increased I SC; the current was reduced by blocking PKA, apical Cl− channels, basolateral NKCC1 (a Na+-K+-2Cl−cotransporter), and basolateral Cl−/HCO[Formula: see text]exchangers. In fluid transport studies, hIMCDi monolayers absorbed fluid in the basal state and forskolin reversed net fluid transport to secretion. In hIMCDt monolayers, basal current was not different from zero and cAMP had no effect on I SC. We conclude that AVP and PGE2stimulate cAMP-dependent Cl− secretion by hIMCDi cells, but not hIMCDt cells, in vitro. We suggest that salt secretion at specialized sites along human collecting ducts may be important in the formation of the final urine.

scholarly journals Antigen-stimulated Activation of Phospholipase D1b by Rac1, ARF6, and PKCα in RBL-2H3 Cells

2002 ◽  
Vol 13 (4) ◽  
pp. 1252-1262 ◽  
Author(s):  
Dale J. Powner ◽  
Matthew N. Hodgkin ◽  
Michael J.O. Wakelam
Keyword(s):  
Plasma Membrane ◽  
Cell Types ◽  
Enzyme Activation ◽  
Dominant Negative ◽  
Ph Domain ◽  
Phosphatidylinositol 3 Kinase ◽  
Stimulation Of ◽  
Phosphatidylinositol 3

Phospholipase D (PLD) activity can be detected in response to many agonists in most cell types; however, the pathway from receptor occupation to enzyme activation remains unclear. In vitro PLD1b activity is phosphatidylinositol 4,5-bisphosphate dependent via an N-terminal PH domain and is stimulated by Rho, ARF, and PKC family proteins, combinations of which cooperatively increase this activity. Here we provide the first evidence for the in vivo regulation of PLD1b at the molecular level. Antigen stimulation of RBL-2H3 cells induces the colocalization of PLD1b with Rac1, ARF6, and PKCα at the plasma membrane in actin-rich structures, simultaneously with cooperatively increasing PLD activity. Activation is both specific and direct because dominant negative mutants of Rac1 and ARF6 inhibit stimulated PLD activity, and surface plasmon resonance reveals that the regulatory proteins bind directly and independently to PLD1b. This also indicates that PLD1b can concurrently interact with a member from each regulator family. Our results show that in contrast to PLD1b's translocation to the plasma membrane, PLD activation is phosphatidylinositol 3-kinase dependent. Therefore, because inactive, dominant negative GTPases do not activate PLD1b, we propose that activation results from phosphatidylinositol 3-kinase–dependent stimulation of Rac1, ARF6, and PKCα.

Direct fibrogenic effects of aldosterone on normotensive kidney: an effect modified by 11β-HSD activity

2010 ◽  
Vol 298 (5) ◽  
pp. F1178-F1187 ◽  
Author(s):  
Andrew S. Brem ◽  
David J. Morris ◽  
Yan Ge ◽  
Lance Dworkin ◽  
Evelyn Tolbert ◽  
...  
Keyword(s):  
Collecting Duct ◽  
Tissue Growth ◽  
Proximal Tubule Cells ◽  
Ru 486 ◽  
Collecting Duct Cells ◽  
Medullary Collecting Duct ◽  
Induced Changes ◽  
Rat Proximal Tubule ◽  
Sirius Red

Aldosterone (Aldo) can be a profibrotic factor in cardiovascular and renal tissues. This study tests the hypothesis that prolonged Aldo exposure is able to directly induce fibrotic changes in the kidney of a normal nonhypertensive animal. Immortalized rat proximal tubule cells (IRPTC) containing 11β-hydroxysteroid dehydrogenase (11β-HSD1) but no mineralocorticoid receptors (MR) and mouse inner medullary collecting duct cells (IMCD) containing 11β-HSD2 and MR were examined. IRPTC exposed to Aldo or corticosterone (10 nM) for 48 h demonstrated no change in collagen production as assessed by Sirius red staining. In contrast, IMCD treated with Aldo exhibited a marked increase in the expression of collagen, fibronectin, and connective tissue growth factor (CTGF), whereas corticosterone alone had no effect. The Aldo-induced overexperession of collagen, fibronectin, and CTGF was substantially attenuated by the MR antagonist RU-318 and by the 11β-HSD end product 11-dehydrocorticosterone, but not by the glucocorticoid receptor antagonist RU-486. In vivo, early fibrotic changes with elevated collagen, fibronectin, and CTGF expression were observed in kidneys isolated from normotensive adrenalectomized mice receiving a continuous infusion of Aldo (8 μg·kg−1·day−1) for 1 wk. These changes were not present in corticosterone-treated mice. Aldo-induced changes were attenuated in adrenally intact mice and in mice treated with RU-318 or 11-dehydrocorticosterone. Thus, extended Aldo exposure produces fibrotic changes in cells containing MR and in normal kidneys. MR antagonists and the end products of 11β-HSD attenuate these fibrogenic effects.

Stimulation of AQP2 membrane insertion in renal epithelial cells in vitro and in vivo by the cGMP phosphodiesterase inhibitor sildenafil citrate (Viagra)

2005 ◽  
Vol 288 (6) ◽  
pp. F1103-F1112 ◽  
Author(s):  
Richard Bouley ◽  
Nuria Pastor-Soler ◽  
Ori Cohen ◽  
Margaret McLaughlin ◽  
Sylvie Breton ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Collecting Duct ◽  
Sildenafil Citrate ◽  
Membrane Insertion ◽  
Vasopressin Receptor ◽  
Pde5 Inhibitors ◽  
Principal Cells ◽  
Medullary Collecting Duct

Vasopressin-stimulated insertion of the aquaporin 2 (AQP2) water channel into the plasma membrane of kidney collecting duct principal cells is a key event in the urinary concentrating mechanism. The paradigm for vasopressin-receptor signaling involves cAMP-mediated protein kinase A activation, which results in the functionally critical phosphorylation of AQP2 on amino acid serine 256. We previously showed that a parallel cGMP-mediated signaling pathway also leads to AQP2 membrane insertion in AQP2-transfected LLC-PK1 (LLC-AQP2) cells and in outer medullary collecting duct principal cells in situ (Bouley R, Breton S, Sun T, McLaughlin M, Nsumu NN, Lin HY, Ausiello DA, and Brown D. J Clin Invest 106: 1115–1126, 2000). In the present report, we show by immunofluorescence microscopy, and Western blotting of plasma membrane fractions, that 45-min exposure of LLC-AQP2 cells to the cGMP phosphodiesterase type 5 (PDE5) inhibitors sildenafil citrate (Viagra) or 4-{[3',4'-methylene-dioxybenzyl]amino}-6-methoxyquinazoline elevates intracellular cGMP levels and results in the plasma membrane accumulation of AQP2; i.e., they mimic the vasopressin effect. Importantly, our data also show that acute exposure to PDE5 inhibitors for 60 min induces apical accumulation of AQP2 in kidney medullary collecting duct principal cells both in tissue slices incubated in vitro as well as in vivo after intravenous injection of Viagra into rats. These data suggest that AQP2 membrane insertion can be induced independently of vasopressin-receptor activation by activating a parallel cGMP-mediated signal transduction pathway with cGMP PDE inhibitors. These results provide proof-of-principle that pharmacological activation of vasopressin-independent, cGMP signaling pathways could aid in the treatment of those forms of nephrogenic diabetes insipidus that are due to vasopressin-2 receptor dysfunction.

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